Difference between revisions of "Team:TokyoTech/Experiment/TraR Reporter Assay"
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Latest revision as of 00:14, 2 November 2017
<!DOCTYPE html>
TraR Reporter Assay
Introduction
In our project, we use a kind of Quorum Sensing signaling molecules (Acyl-homoserine lactones, AHLs), 3OC8HSL (hereafter C8) as a signaling molecule of cross-kingdom communication from bacteria to human cells (Read TraI Assay page). Therefore, we searched the way to detect that C8 was synthesized by E. coli introduced C8 synthesis gene, from traI. This section describes the evaluation of Tra system (a kind of Quorum Sensing derived from Agrobacterium tumefaciens).
Summary
The purpose of the experiment is to evaluate the reporter gene of Tra system, from traR works correctly in E. coli cells. We constructed the two plasmids shown below and introduced them to E. coli. To the end, adding C8 into the E. coli and investigate TraR-C8 complex activate transcription from Ptra.
- Plasmids
Sample(Fig. 1)
A. Ptet – rbs – traR (pSB6A1)
B. Ptra – rbs – gfp (pSB3K3)
Negative control (Fig. 2)
C. pSB6A1
D. pSB3K3
Results
The results showed that the value of RFU of GFP / Turbidity of samples and that of negative controls was same. This indicated that GFP expression was not induced by TraR-C8 complex.
Discussion
From the results, Tra reporter system did not work in E. coli. This is because the tra box where TraR-C8 complex binds upstream Ptra is essential to active transcription(Read Chimeric Transcription Factor Assay page) and the number of this region is not enough for non-A. tumefaciens organisms to activate transcription. From other our experiment, C8 could be detect by Lux system (a kind of Quorum Sensing system derived from Vibrio fischeri). Thus, we decided to use Lux system to detect that C8 was synthesized by E. coli introduced traI (Read TraI Assay page)
Reference
[1] Foundational Platform for Mammalian Synthetic Biology, 2012 Noah Davidsohn et.al
[2]Conserved cis-Acting Promoter Elements Are Required for Density-Dependent Transcription of Agrobacterium tumefaciens Conjugal Transfer Genes, 1995 CLAY FUQUA et.al
